Benzothiazole or benzoxazole compounds as SUMO activators

ABSTRACT

Provided are SUMO activators, which can enhance SUMOylation of SERCA2a, which are useful in the treatment of heart failure, cardiovascular diseases, cancer, neurodegenerative disorders, viral infection, bacterial infection, liver disease, inflammation, and other diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/424,289 filed Feb. 26, 2015, which is a § 371 National StageApplication of PCT/US2013/057264, filed Aug. 29, 2013, which claimspriority to U.S. provisional application No. 61/694,667 filed Aug. 29,2012, each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present application relates to SUMO activators, which can enhanceSUMOylation of SERCA2a, which are useful in the treatment of heartfailure, cardiovascular diseases, cancer, neurodegenerative disorders,viral infection, bacterial infection, liver disease, inflammation, andother diseases.

BACKGROUND

Heart failure (HF) remains a leading cause of death in Western countriesand the development of new therapeutic agents for HF has beenchallenged. The recent major advances in the understanding of molecularsignaling in the cardiac myocytes under the pathological stress hassuggested the way for different approaches to treating heart disease, inparticular to regulate intrinsic targets on the intracellular side. Thecalcium-transporting ATPase ATP2A2 (SERCA2a) is ATPase responsible forCa²⁺ re-uptake during excitation-contraction coupling. A characteristicof heart failure is impaired Ca²⁺ uptake resulting from decreasedexpression and reduced activity of SERCA2a. To this end, restoration ofSERCA2a expression by gene transfer can be effective in improvingcardiac function in animal models and heart-failure patients. It wasfound that the levels and activity of SERCA2a in cardiac myocytes aremodulated by small ubiquitin-like modifier type 1 (SUMO I)-mediatedunique post-translational modification (PTM), named SUMOylation (Kho C,Lee A, Jeong D, Oh J G Chaanine A H, Kizana E, Park W J, Hajjar R J,“SUMO1-dependent modulation of SERCA2a in heart failure”, Nature 2011Sep. 7; 477(7366):601-5). SERCA2a is SUMOYLated by SUMO1 at two specificsites Lysine 480 and 585. The levels of SUMO1 and the SUMOylation ofSERCA2a itself were greatly reduced in failing hearts. SUMO1 restitutionby adeno-associated-virus-mediated gene delivery maintained the proteinabundance of SERCA2a and markedly improved cardiac function in mice withheart failure. This effect was comparable to SERCA2A gene delivery.Since it has been shown that SUMO1 enhances the stability and the ATPaseactivity of SERCA2a, its decrease causes further dysfunction of SERCA2aand further worsening of dysfunction. Further, gain of functionexperiments by transgenesis and gene therapy showed that SUMO1 genetherapy rescues contractile function and improves mortality in models ofheart failure. To this end, there is a need to develop new smallmolecules that increase SERCA2a SUMOylation, which are are useful fortreating HF.

Further, induction of SUMOylation has also been implicated in thetreatment of cancer (Kira Bettermann, Martin Benesch, Serge Weis,Johannes Haybaeck. SUMOylation in carcinogenesis. Cancer Letters (2012)316, 113-125), neurodegenerative disorders such as Huntington's disease(Steffan, J. S. et al. SUMO modification of Huntingtin and Huntington'sdisease pathology. Science (2004) 304, 100-104), Parkinson's disease(Dorval, V., Fraser, P. E. Small ubiquitin-like modifier (SUMO)modification of natively unfolded proteins tau and a-synuclein. J. Biol.Chem. (2006) 281, 9919-9924), Alzheimer's disease (Zhang, Y Q. andSarge, K. D. Sumoylation of amyloid precursor protein negativelyregulates Ab aggregate levels. (2008) Biochem. Biophys. Res. Commun.374, 673-678), and amyotrophic lateral sclerosis (ALS) (Fei, E. et al.SUMO-1 modification increases human SOD1 stability and aggregation.Biochem. Biophys. Res. Commun. (2006) 347, 406-412), viral and bacterialinfection (Békés M, Drag M. Trojan horse strategies used by pathogens toinfluence the small ubiquitin-like modifier (SUMO) system of hosteukaryotic cells. J Innate Immun. (2012) 4, 159-67), liver disease (GuoW H, Yuan L H, Xiao Z H, Liu D, Zhang J X. Overexpression of SUMO-1 inhepatocellular carcinoma: a latent target for diagnosis and therapy ofhepatoma. J Cancer Res Clin Oncol. (2011) 137, 533-41), and inflammation(Pascual G, Fong A L, Ogawa S, Gamliel A, Li A C, Perissi V, Rose D W,Willson T M, Rosenfeld M G, Glass C K. A SUMOylation-dependent pathwaymediates transrepression of inflammatory response genes by PPAR-gamma.Nature (2005) 437, 759-63).

To this end, there is a need to develop new small molecules thatincrease SERCA2a SUMOylation, which are useful for treating HF. Thisapplication addresses this need and others.

SUMMARY

The present application provides, inter alia, a method of treating heartfailure, cardiac hypertrophy, myocarditis, myocardial infarction,ischemia, cardiac arrhythmias, vascular rhexis, cardiac arrhythmia,valvulopathy, diastolic dysfunction, hypertension, cancer,neurodegenerative disorders, viral infection, bacterial infection, liverdisease, or inflammation in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of acompound of Formula II:

or a pharmaceutically acceptable salt thereof; wherein:

Ar is aryl or heteroaryl, each of which is optionally substituted with1, 2, 3, 4, 5, or 6 independently selected R^(1a) groups;

W is S or O;

Y is S or O;

each X is independently CH or N;

Z is O, S, or NR^(A);

NR^(A) is H or C₁₋₄ alkyl;

R¹, R², R³, and R⁴ are each independently selected from hydrogen, halo,CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are eachoptionally substituted with 1, 2, or 3 groups independently selectedfrom halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino; and

each R^(1a) is independently selected from halo, CN, nitro, hydroxy,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl,C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl,C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino,di-C₁₋₄-alkylamino, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆alkylcarbonyl are each optionally substituted with 1, 2, or 3 groupsindependently selected from halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃alkylamino, and di-C₁₋₃-alkylamino; or

two adjacent R^(1a) taken together with the atoms to which they areattached can form a 3-7 membered carbocyclic or 4-6 memberedheterocyclic ring, each of which is optionally substituted with 1, 2, 3,or 4 C₁₋₃ alkyl groups.

In some embodiments, heart failure is selected from congestive heartfailure (CHF), chronic heart failure, and ischemic heart failure.

The present invention further provides a method of treating heartfailure, cardiac hypertrophy, myocarditis, myocardial infarction,ischemia, cardiac arrhythmias, vascular rhexis, cardiac arrhythmia,valvulopathy, diastolic dysfunction, hypertension, cancer,neurodegenerative disorders, viral infection, bacterial infection, liverdisease, or inflammation in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of acompound of a compound of Formula I:

or a pharmaceutically acceptable salt thereof; wherein:

Ar is aryl or heteroaryl, each of which is optionally substituted with1, 2, 3, 4, 5, or 6 independently selected R^(1a) groups;

W is S or O;

Y is S or O;

Z is O, S, or NR^(A);

NR^(A) is H or C₁₋₄ alkyl;

R¹, R², R³, and R⁴ are each independently selected from hydrogen, halo,CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are eachoptionally substituted with 1, 2, or 3 groups independently selectedfrom halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino; and

each R^(1a) is independently selected from halo, CN, nitro, hydroxy,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl,C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl,C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino,di-C₁₋₄-alkylamino, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆alkylcarbonyl are each optionally substituted with 1, 2, or 3 groupsindependently selected from halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃alkylamino, and di-C₁₋₃-alkylamino.

In some embodiments, Ar is phenyl, which is optionally substituted by 1,2, 3, 4, or 5 independently selected R^(1a) groups.

In some embodiments, each R^(1a) is independently selected from halo,CN, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆haloalkoxy. In some embodiments, each R^(1a) is independently selectedfrom hydrogen and C₁₋₆ alkoxy.

In some embodiments, W is S.

In some embodiments, Y is O.

In some embodiments, Z is NH.

In some embodiments, R¹, R², R³, and R⁴ are independently selected fromhydrogen, halo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆ alkylamino, and di-C₁₋₄-alkylamino.In some embodiments, R¹, R², R³, and R⁴ are independently selected fromhydrogen, halo, and C₁₋₆ alkoxy. In some embodiments, at least three ofR¹, R², R³, and R⁴ are hydrogen. In some embodiments, R¹, R², R³, and R⁴are hydrogen.

In some embodiments, the compound is a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula Ib:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula Ic:

or a pharmaceutically acceptable salt thereof; wherein:

R^(2a), R^(2b), R^(2c), R^(2d), and R^(2e) are each independentlyselected from hydrogen, halo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl, C₂₋₆heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl, aminosulfonylamino, C₁₋₆alkylaminosulfonylamino, and di-C₁₋₄ alkylaminosulfonylamino; whereinsaid C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy,C₁₋₆ haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are eachoptionally substituted with 1, 2, or 3 groups independently selectedfrom halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino.

In some embodiments, R¹, R², R³, R⁴, R^(2a), R^(2b), R^(2c), R^(2d), andR^(2e) are each independently selected from hydrogen, halo, CN, hydroxy,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy.

In some embodiments, each X is CH.

In some embodiments, the compound is a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIb:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIc:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound selected from thecompounds of Table 1a, Table 1b, Table 1c, and Table 1d; or apharmaceutically acceptable salt thereof.

Non-limiting examples of compounds of Formula II include:

-   5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   5-(2,4-dimethoxyphenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-phenyl-1,3,4-oxadiazol-2-amine;-   N-(6-methoxybenzo[d]thiazol-2-yl)-5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(6-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   5-(4-chlorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   5-(2-chlorophenyl)-N-(6-fluorobenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   N-(6-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine;-   N-(6-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(6-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(5-methoxybenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(5-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   5-(4-methoxyphenyl)-N-(6-(methylthio)benzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;    and-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine,    or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from:

-   N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;    and-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine,    or a pharmaceutically acceptable salt thereof.

The present application also provides a method which further comprisesadministering to the patient an adeno-associated vector (AAV) comprisingSERCA2a.

The present application further provides a method of activating SUMO1,comprising contacting comprising contacting a cell with a compound,salt, or composition described herein, in an amount effective toactivate SUMO1.

The present application further provides a compound or salt as describedherein for use in any of the methods described herein.

The present application further provides use of a compound or salt asdescribed herein for manufacture of a medicament for use in any of themethod described herein.

The present application further provides a pharmaceutical compositioncomprising any of the compounds described herein, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier. The present application further provides a compound, which isN-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine,or a pharmaceutically acceptable salt thereof.

DESCRIPTION OF DRAWINGS

FIG. 1A depicts intracellular fluorescence measurements for DMSO controlversus Example 1 in HEK-293 cells expressing YFP-SUMO1 andpcDNA3.0-SERCA2a. FIG. 1B depicts immunoblotting from cells treated withExample 1 or DMSO.

FIG. 2A depicts effects of Example 1 on the mechanical properties of theARVMs from measurements using a video-based edge detection system(IonOptix). FIG. 2B depicts SERCA2a SUMOylation profiling in the sameset of ARVMs.

FIG. 3A depicts dose-dependent effects of Example 1 of a SERCA2aSUMOylation activator.

FIG. 3B depicts time-dependent effects of Example 1 of a SERCA2aSUMOylation activator.

FIG. 4A depicts a plot of ATP hydrolysis vs. Example 1 concentration inthe presence of SUMO-1.

FIG. 4B depicts a blot of concentrations of Example 1 and a SUMO-1 andSUMO E1 enzyme

FIG. 4C depicts a blot of transfer of the activated SUMO-1 from SUMO E1to the unique SUMO E2 enzyme.

FIG. 4D depicts computer modeling of the EC₅₀ of PPi, E1-SUMO-1, andE2-SUMO1.

FIG. 5A depicts time-dependent effects of Example 1 in mouse models.

FIG. 5B depicts dose-dependent effects of Example 1 in mouse models.

FIG. 6A depicts effects of Example 1 on hemodynamic parameters in aSerca2 knock-out mouse model.

FIG. 6B depicts dose-dependent enhancement of cardiac contractility ofExample 1 in a Serca2 knock-out mouse model.

FIG. 7A depicts the compound screening strategy used to identify Example1 that targets the SERCA2a SUMOylation.

FIG. 7B depicts cellular imaging of HEK-293 cells with Example 1 at 10μM concentration.

FIG. 7C depicts a blot of the effect of Example 1 on increasing SERCA2aSUMOylation in HEK-293 cells.

FIG. 8A depicts the functional analysis of Example 1 compared to othercompounds identified from the screening strategy.

FIG. 8B depicts endogenous SERCA2a SUMOylation status for 8 compoundsand a vehicle.

FIG. 9A depicts the characterization of TAC mice used for in vivostudies.

FIG. 9B depicts the effect of Example 1 on hemodynamic parameters in TACmice.

FIG. 9C depicts dobutamine response as a function of dP/dt max vs.ng/BWg in TAC mice and a sham.

FIG. 10A depicts the effects of vehicle on hemodynamic function in shamanimals. FIG. 10B depicts the effects of Example 1 on hemodynamicfunction in sham animals.

FIG. 11A depicts the animal characterization of Serca2 knockout mice.

FIG. 11B depicts a bar graph and blot of control WT and Serca2 knockoutmice.

FIG. 12 depicts the pharmacokinetic analysis of the compound of Example1 in mice at a 10 mg/kg intraperitoneal dose.

FIG. 13 depicts the effects of Example 1 in a cancer cell line panel.

FIG. 14 depicts the effects of Examples 1-4 on YFP-SUMO1 accumulation inthe nucleus and SUMOylation of SERCA2a.

FIG. 15 depicts the effects of Example 1 on SERCA2a SUMOylation atvarying doses.

FIG. 16 depicts the protocol for hemodynamic study.

FIG. 17 depicts the pressure-volume relationships in TAC mice withincreasing concentrations of vehicle.

FIG. 18 depicts the pressure-volume relationships in TAC mice withincreasing concentrations of Example 1.

FIG. 19 depicts the effects of Example 1 on various hemodynamicparameters.

FIG. 20 depicts the effects of Example 1 on contractility in a pig asdetermined by dP/dt.

DETAILED DESCRIPTION

The present application provides, inter alia, a method of treating heartfailure, cardiac hypertrophy, myocarditis, myocardial infarction,ischemia, cardiac arrhythmias, vascular rhexis, cardiac arrhythmia,valvulopathy, diastolic dysfunction, hypertension, cancer,neurodegenerative disorders, viral infection, bacterial infection, liverdisease, or inflammation in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of acompound of Formula II:

or a pharmaceutically acceptable salt thereof; wherein:

Ar is aryl or heteroaryl, each of which is optionally substituted with1, 2, 3, 4, 5, or 6 independently selected R^(1a) groups;

W is S or O;

Y is S or O;

each X is independently CH or N;

Z is O, S, or NR^(A);

NR^(A) is H or C₁₋₄ alkyl;

R¹, R², R³, and R⁴ are each independently selected from hydrogen, halo,CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are eachoptionally substituted with 1, 2, or 3 groups independently selectedfrom halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino; and

each R^(1a) is independently selected from halo, CN, nitro, hydroxy,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl,C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl,C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino,di-C₁₋₄-alkylamino, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆alkylcarbonyl are each optionally substituted with 1, 2, or 3 groupsindependently selected from halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃alkylamino, and di-C₁₋₃-alkylamino; or

two adjacent R^(1a) taken together with the atoms to which they areattached can form a 3-7 membered carbocyclic or 4-6 memberedheterocyclic ring, each of which is optionally substituted with 1, 2, 3,or 4 C₁₋₃ alkyl groups.

In some embodiments, the present application provides, a method oftreating heart failure, cardiac hypertrophy, myocarditis, myocardialinfarction, ischemia, cardiac arrhythmias, vascular rhexis, cardiacarrhythmia, valvulopathy, diastolic dysfunction, hypertension, cancer,neurodegenerative disorders, viral infection, bacterial infection, liverdisease, or inflammation in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof; wherein:

Ar is aryl or heteroaryl, each of which is optionally substituted with1, 2, 3, 4, 5, or 6 independently selected R^(1a) groups;

W is S or O;

Y is S or O;

Z is O, S, or NR^(A);

NR^(A) is H or C₁₋₄ alkyl;

R¹, R², R³, and R⁴ are each independently selected from hydrogen, halo,CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are eachoptionally substituted with 1, 2, or 3 groups independently selectedfrom halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino; and

each R^(1a) is independently selected from halo, CN, nitro, hydroxy,C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl,C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl,C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino,di-C₁₋₄-alkylamino, C₁₋₆ alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆alkylcarbonyl are each optionally substituted with 1, 2, or 3 groupsindependently selected from halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃alkylamino, and di-C₁₋₃-alkylamino.

In some embodiments, the heart failure is selected from congestive heartfailure (CHF), chronic heart failure, and ischemic heart failure.

Cancers include, but are not limited to, solid tumors such as breast,ovarian, prostate, lung, kidney, gastric, colon, testicular, head andneck, pancreas, brain, melanoma, and other tumors of tissue organs andcancers of the blood cells, such as lymphomas and leukemias, includingacute myelogenous leukemia, chronic lymphocytic leukemia, T celllymphocytic leukemia, and B cell lymphomas.

Inflammatory disorders include, but are not limited to, transplantrejection, including skin graft rejection; chronic inflammatorydisorders of the joints, including arthritis, rheumatoid arthritis,osteoarthritis and bone diseases associated with increased boneresorption; inflammatory bowel diseases such as ileitis, ulcerativecolitis, Barrett's syndrome, and Crohn's disease; inflammatory lungdisorders such as asthma, adult respiratory distress syndrome, andchronic obstructive airway disease; inflammatory disorders of the eyeincluding corneal dystrophy, trachoma, onchocerciasis, uveitis,sympathetic ophthalmitis and endophthalmitis; chronic inflammatorydisorders of the gums, including gingivitis and periodontitis;tuberculosis; leprosy; inflammatory diseases of the kidney includinguremic complications, glomerulonephritis and nephrosis; inflammatorydisorders of the skin including sclerodermatitis, psoriasis and eczema;inflammatory diseases of the central nervous system, including chronicdemyelinating diseases of the nervous system, multiple sclerosis,AIDS-related neurodegeneration and Alzheimer's disease, infectiousmeningitis, encephalomyelitis, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis and viral or autoimmuneencephalitis; autoimmune disorders, immune-complex vasculitis, systemiclupus and erythematodes; systemic lupus erythematosus (SLE); andinflammatory diseases of the heart such as cardiomyopathy, ischemicheart disease hypercholesterolemia, atherosclerosis; as well as variousother diseases with significant inflammatory components, includingpreeclampsia; chronic liver failure, brain and spinal cord trauma, andcancer. There may also be a systemic inflammation of the body,exemplified by gram-positive or gram negative shock, hemorrhagic oranaphylactic shock, or shock induced by cancer chemotherapy in responseto pro-inflammatory cytokines, e.g., shock associated withpro-inflammatory cytokines. Such shock can be induced, e.g., by achemotherapeutic agent used in cancer chemotherapy.

Neurodegenerative disorders include, but are not limited to Huntington'sdisease, Parkinson's disease, Alzheimer's disease, and amyotrophiclateral sclerosis (ALS).

Viral infections, include but are not limited to, infections by ahepatitis virus (e.g., hepatitis B or C), human immunodeficiency virus(HIV), rhinovirus, herpes-zoster virus (VZV), herpes simplex virus(e.g., HSV-1 or HSV-2), cytomegalovirus (CMV), vaccinia virus, influenzavirus, encephalitis virus, hantavirus, arbovirus, West Nile virus, humanpapilloma virus (HPV), Epstein-Ban virus, and respiratory syncytialvirus.

Liver diseases include, but are not limited to liver cirrhosis,alcoholic liver cirrhosis, fatty liver, toxipathic liver diseases, andacute and chronic hepatitis.

In some embodiments, Ar is phenyl, which is optionally substituted by 1,2, 3, 4, or 5 independently selected R^(1a) groups.

In some embodiments, each R^(1a) is independently selected from halo,CN, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, and C₁₋₆haloalkoxy.

In some embodiments, each R^(1a) is independently selected from hydrogenand C₁₋₆ alkoxy.

In some embodiments, W is S.

In some embodiments, Y is O.

In some embodiments, Z is NH.

In some embodiments, R¹, R², R³, and R⁴ are independently selected fromhydrogen, halo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆ alkylamino, and di-C₁₋₄-alkylamino.

In some embodiments, R¹, R², R³, and R⁴ are independently selected fromhydrogen, halo, and C₁₋₆ alkoxy.

In some embodiments, at least three of R¹, R², R³, and R⁴ are hydrogen.

In some embodiments, R¹, R², R³, and R⁴ are hydrogen.

In some embodiments, the compound is a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula Ib:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula Ic:

or a pharmaceutically acceptable salt thereof; wherein:

R^(2a), R^(2b), R^(2c), R^(2d), and R^(2e) are each independentlyselected from hydrogen, halo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl, C₂₋₆heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl, aminosulfonylamino, C₁₋₆alkylaminosulfonylamino, and di-C₁₋₄ alkylaminosulfonylamino; whereinsaid C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆ alkoxy,C₁₋₆ haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆alkylcarbamyl, di(C₁₋₄ alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are eachoptionally substituted with 1, 2, or 3 groups independently selectedfrom halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, anddi-C₁₋₃-alkylamino.

In some embodiments of the compounds of Formula Ic, R¹, R², R³, R⁴,R^(2a), R^(2b), R^(2c), R^(2d), and R^(2e) are each independentlyselected from hydrogen, halo, CN, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ alkoxy, and C₁₋₆ haloalkoxy.

In some embodiments, X is CH. In some embodiments, X is N.

In some embodiments, the compound is a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is a compound of Formula IIb:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIc:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIIa:

or a pharmaceutically acceptable salt thereof.

Some examples of Formula IIIa may be synthesized using the followingsynthetic sequence:

In some embodiments, the compound is a compound of Formula IIIb:

or a pharmaceutically acceptable salt thereof.

Some examples of Formula IIIb may be synthesized using the followingsynthetic sequence:

In some embodiments, the compound is a compound of Formula IIIc:

or a pharmaceutically acceptable salt thereof.

Some examples of Formula IIIc may be synthesized using the followingsynthetic sequence:

The present invention further provides a compounds as described herein,or a pharmaceutically acceptable salt thereof.

Additional prophetic compounds of Formula II are shown in Table 1a,Table 1b, Table 1c, and Table 1d.

TABLE 1a Oxadiazoles.

TABLE 1b Thiadiazoles.

TABLE 1c Thiophenes.

TABLE 1d Benzoxazoles.

In some embodiments, the compound is a compound of Table 1a, Table 1b,Table 1c, or Table 1d, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from:

-   5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   5-(2,4-dimethoxyphenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-phenyl-1,3,4-oxadiazol-2-amine;-   N-(6-methoxybenzo[d]thiazol-2-yl)-5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(6-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   5-(4-chlorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   5-(2-chlorophenyl)-N-(6-fluorobenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   N-(6-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine;-   N-(6-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(6-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(5-methoxybenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(5-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   5-(4-methoxyphenyl)-N-(6-(methylthio)benzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine;-   N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;    and-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine,    or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from:

-   N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine;-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine;    and-   N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine,    or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound isN-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine,or pharmaceutically acceptable salt thereof.

The compounds described herein can activate SUMO1. Accordingly, thepresent application further provides a method of activating SUMO1,comprising contacting comprising contacting a cell with a compound,salt, or composition described herein, in an amount effective toactivate SUMO1. The contacting can be done in vivo or in vitro. Infurther embodiments, the compounds of the present application can beused to activate SUMO1 in an individual in need of the activation byadministering a compound, salt, or composition described herein, in anamount effective to activate SUMO1.

The present application further provides a pharmaceutical compositioncomprising any of the compounds described herein, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier.

The present application further provides any of the compounds describedherein, or a pharmaceutically acceptable salt thereof.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment (while theembodiments are intended to be combined as if written in multiplydependent form). Conversely, various features of the invention whichare, for brevity, described in the context of a single embodiment, canalso be provided separately or in any suitable subcombination.

At various places in the present specification, substituents ofcompounds of the invention are disclosed in groups or in ranges. It isspecifically intended that the invention include each and everyindividual subcombination of the members of such groups and ranges. Forexample, the term “C₁₋₆ alkyl” is specifically intended to individuallydisclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is to beunderstood that substitution at a given atom is limited by valency.

As used herein, the term “C_(n-m) alkyl”, employed alone or incombination with other terms, refers to a saturated hydrocarbon groupthat may be straight-chain or branched, having n to m carbon atoms. Insome embodiments, the alkyl group contains 1 to 6, 1 to 4 or 1 to 3carbon atoms. Examples of alkyl moieties include, but are not limitedto, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl,n-hexyl, 1,2,2-trimethylpropyl, and the like.

As used herein, “C_(n-m) alkenyl” refers to an alkyl group having one ormore double carbon-carbon bonds and having n to m carbons. In someembodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3carbon atoms. Example alkenyl groups include, but are not limited to,ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

As used herein, “C_(n-m) alkynyl” refers to an alkyl group having one ormore triple carbon-carbon bonds and having n to m carbons. Examplealkynyl groups include, but are not limited to, ethynyl, propyn-1-yl,propyn-2-yl, and the like. In some embodiments, the alkynyl moietycontains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkoxy”, employed alone or incombination with other terms, refers to a group of formula —O-alkyl,wherein the alkyl group has n to m carbons. Example alkoxy groupsinclude methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy),t-butoxy, and the like. In some embodiments, the alkyl group has 1 to 6or 1 to 4 carbon atoms.

As used herein, the term “amino” refers to a group of formula —NH₂.

As used herein, the term “C_(n-m) alkylamino” refers to a group offormula —NH(alkyl), wherein the alkyl group has n to m carbon atoms. Insome embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.

As used herein, the term “di-C_(n-m)-alkylamino” refers to a group offormula —N(alkyl)₂, wherein the two alkyl groups each has,independently, n to m carbon atoms. In some embodiments, each alkylgroup independently has 1 to 6 or 1 to 4 carbon atoms.

As used herein, the term “C_(n-m) alkoxycarbonyl” refers to a group offormula —C(O)O-alkyl, wherein the alkyl group has n to m carbon atoms.In some embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.

As used herein, the term “C_(n-m) alkylcarbonyl” refers to a group offormula —C(O)— alkyl, wherein the alkyl group has n to m carbon atoms.In some embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.

As used herein, the term “C_(n-m) alkylsulfonyl” refers to a group offormula —S(O)₂-alkyl, wherein the alkyl group has n to m carbon atoms.In some embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.

As used herein, the term “C_(n-m) alkylcarbonyloxy” refers to a group offormula —OC(O)-alkyl, wherein the alkyl group has n to m carbon atoms.In some embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.

As used herein, the term “C_(n-m) alkylcarbonylamino” refers to a groupof formula —NHC(O)-alkyl, wherein the alkyl group has n to m carbonatoms. In some embodiments, the alkyl group has 1 to 6 or 1 to 4 carbonatoms.

As used herein, the term “carbamyl” refers to a group of formula—C(O)—NH₂.

As used herein, the term “C_(n-m) alkylcarbamyl” refers to a group offormula —C(O)—NH(alkyl), wherein the alkyl group has n to m carbonatoms. In some embodiments, the alkyl group has 1 to 6 or 1 to 4 carbonatoms.

As used herein, the term “di(C_(n-m)-alkyl)carbamyl” refers to a groupof formula —C(O)N(alkyl)₂, wherein the two alkyl groups each has,independently, n to m carbon atoms. In some embodiments, each alkylgroup independently has 1 to 6 or 1 to 4 carbon atoms.

As used herein, the term “C₁₋₄ alkylsulfonylamino” refers to a group offormula —NHS(O)₂-alkyl, wherein said alkyl has 1 to 4 carbon atoms.

As used herein, the term “aminosulfonyl” refers to a group of formula—S(O)₂NH₂.

As used herein, the term “C₁₋₄ alkylaminosulfonyl” refers to a group offormula —S(O)₂NH(alkyl), wherein said alkyl has 1 to 4 carbon atoms.

As used herein, the term “di-C₁₋₄ alkylaminosulfonyl” refers to a groupof formula —S(O)₂N(alkyl)₂, wherein each alkyl independently has 1 to 4carbon atoms.

As used herein, the term “aminosulfonylamino” refers to a group offormula —NHS(O)₂NH₂, wherein said alkyl has 1 to 4 carbon atoms.

As used herein, the term “C₁₋₄ alkylaminosulfonylamino,” refers to agroup of formula —NHS(O)₂NH(alkyl), wherein said alkyl has 1 to 4 carbonatoms.

As used herein, the term “di-C₁₋₄ alkylaminosulfonylamino” refers to agroup of formula —NHS(O)₂N(alkyl)₂, wherein each alkyl independently has1 to 4 carbon atoms.

As used herein, “halo” or “halogen”, employed alone or in combinationwith other terms, includes fluoro, chloro, bromo, and iodo.

As used herein, the term “C_(n-m) haloalkyl”, employed alone or incombination with other terms, refers to an C_(n-m) alkyl group having upto {2(n to m)+1} halogen atoms which may either be the same ordifferent. In some embodiments, the halogen atoms are fluoro atoms. Insome embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.Example haloalkyl groups include CF₃, C₂F₅, CHF₂, CCl₃, CHCl₂, C₂Cl₅,and the like. In some embodiments, the haloalkyl group is a fluoroalkylgroup.

As used herein, “C_(n-m) haloalkoxy” refers to a group of formula—O-haloalkyl having n to m carbon atoms. An example haloalkoxy group isOCF₃. In some embodiments, the haloalkoxy group is fluorinated only. Insome embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.

As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbonsincluding cyclized alkyl and/or alkenyl groups. Cycloalkyl groups caninclude mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groupsand spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, or 7ring-forming carbons (C₃-7). Ring-forming carbon atoms of a cycloalkylgroup can be optionally substituted by oxo or sulfido. Cycloalkyl groupsalso include cycloalkylidenes. Example cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl,norbornyl, norpinyl, norcarnyl, and the like. In some embodiments,cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Alsoincluded in the definition of cycloalkyl are moieties that have one ormore aromatic rings fused (i.e., having a bond in common with) to thecycloalkyl ring, for example, benzo or thienyl derivatives ofcyclopentane, cyclohexane, and the like. A cycloalkyl group containing afused aromatic ring can be attached through any ring-forming atomincluding a ring-forming atom of the fused aromatic ring.

As used herein, “heteroaryl” refers to a monocyclic or polycyclicaromatic heterocycle having at least one heteroatom ring member selectedfrom sulfur, oxygen, and nitrogen. In some embodiments, the heteroarylring has 1, 2, 3, or 4 heteroatom ring members independently selectedfrom nitrogen, sulfur and oxygen. In some embodiments, any ring-formingN in a heteroaryl moiety can be an N-oxide. In some embodiments, theheteroaryl has 5-10 ring atoms and 1, 2, 3 or 4 heteroatom ring membersindependently selected from nitrogen, sulfur and oxygen. In someembodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatomring members independently selected from nitrogen, sulfur and oxygen. Insome embodiments, the heteroaryl is a five-membered or six-memberedheteroaryl ring.

A five-membered heteroaryl ring is a heteroaryl with a ring having fivering atoms wherein one or more (e.g., 1, 2, or 3) ring atoms areindependently selected from N, O, and S. Exemplary five-membered ringheteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl,oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl,tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl,1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

A six-membered heteroaryl ring is a heteroaryl with a ring having sixring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms areindependently selected from N, O, and S. Exemplary six-membered ringheteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl andpyridazinyl.

A “bicyclic C₉₋₁₀ heteroaryl” is bicyclic fused heteroaryl having 9 to10 ring members.

As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic orpolycyclic heterocycles having one or more ring-forming heteroatomsselected from O, N, or S. Included in heterocycloalkyl are monocyclic4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkylgroups can also include spirocycles. Example heterocycloalkyl groupsinclude pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl,tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino,piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, andthe like. Ring-forming carbon atoms and heteroatoms of aheterocycloalkyl group can be optionally substituted by oxo or sulfido(e.g., C(O), S(O), C(S), or S(O)₂, etc.). The heterocycloalkyl group canbe attached through a ring-forming carbon atom or a ring-formingheteroatom. In some embodiments, the heterocycloalkyl group contains 0to 3 double bonds. In some embodiments, the heterocycloalkyl groupcontains 0 to 2 double bonds. Also included in the definition ofheterocycloalkyl are moieties that have one or more aromatic rings fused(i.e., having a bond in common with) to the cycloalkyl ring, forexample, benzo or thienyl derivatives of piperidine, morpholine,azepine, etc. A heterocycloalkyl group containing a fused aromatic ringcan be attached through any ring-forming atom including a ring-formingatom of the fused aromatic ring. In some embodiments, theheterocycloalkyl has 4-10, 4-7 or 4-6 ring atoms with 1 or 2 heteroatomsindependently selected from nitrogen, oxygen or sulfur and having one ormore oxidized ring members.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent invention that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically inactive startingmaterials are known in the art, such as by resolution of racemicmixtures or by stereoselective synthesis. Many geometric isomers ofolefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms.

Compounds of the present application also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond together with the concomitant migration of aproton. Tautomeric forms include prototropic tautomers which areisomeric protonation states having the same empirical formula and totalcharge. Example prototropic tautomers include ketone-enol pairs,amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, andannular forms where a proton can occupy two or more positions of aheterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present application can also include all isotopes ofatoms occurring in the intermediates or final compounds. Isotopesinclude those atoms having the same atomic number but different massnumbers. For example, isotopes of hydrogen include tritium anddeuterium.

The term, “compound,” as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted. Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.hydrates and solvates) or can be isolated.

In some embodiments, the compounds of the present application, or saltsthereof, are substantially isolated. By “substantially isolated” ismeant that the compound is at least partially or substantially separatedfrom the environment in which it was formed or detected. Partialseparation can include, for example, a composition enriched in thecompounds of the present application. Substantial separation can includecompositions containing at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 97%, or at least about 99% by weight of the compounds ofthe present application, or salt thereof. Methods for isolatingcompounds and their salts are routine in the art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The present application also includes pharmaceutically acceptable saltsof the compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present application include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present application can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, non-aqueous media like ether, ethylacetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) oracetonitrile (ACN) are preferred. Lists of suitable salts are found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2(1977), each of which is incorporated herein by reference in itsentirety.

Combination Therapy

In some embodiments, a compound of the present application, or apharmaceutically acceptable salt thereof, can be used in combinationwith another therapeutic agent to treat diseases such as cancer and/orneurological disorders. For example, the additional agent can be atherapeutic agent that is art-recognized as being useful to treat thedisease or condition being treated by the compound of the presentapplication. The additional agent also can be an agent that imparts abeneficial attribute to the therapeutic composition (e.g., an agent thataffects the viscosity of the composition).

The combination therapy contemplated by the invention includes, forexample, administration of a compound of the present application, or apharmaceutically acceptable salt thereof, and additional agent(s) in asingle pharmaceutical formulation as well as administration of acompound of the present application, or a pharmaceutically acceptablesalt thereof, and additional agent(s) in separate pharmaceuticalformulations. In other words, co-administration shall mean theadministration of at least two agents to a subject so as to provide thebeneficial effects of the combination of both agents. For example, theagents may be administered simultaneously or sequentially over a periodof time.

The additional therapeutic agent can be any therapeutic agent useful forthe treatment of the disease states of the methods described herein. Theadditional therapeutic agent can be administered simultaneously orsequentially. In another embodiment, the method further comprisesadministering to the patient a viral expression vector comprisingSERCA2a. In some embodiments, the method further comprises administeringto the patient an adeno-associated vector (AAV) comprising SERCA2a. Forexample, vectors useful in the present methods include, but are notlimited to those described in US 2011/0256101, which is incorporatedherein by reference in its entirety.

In one embodiment, SERCA2 is incorporated into a viral vector to mediatetransfer to a cell. Alternatively, a retrovirus, bovine papilloma virus,an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyomavirus, or an infective virus may be used. Similarly, nonviral methodswhich include, but are not limited to, direct delivery of DNA such as byperfusion, naked DNA transfection, liposome mediated transfection,encapsulation, and receptor-mediated endocytosis may be employed. Thesetechniques are well known to those of skill in the art, and theparticulars thereof do not lie at the crux of the present invention andthus need not be exhaustively detailed herein. For example, a viralvector is used for the transduction of pulmonary cells to deliver atherapeutically significant polynucleotide to a cell. The virus may gainaccess to the interior of the cell by a specific means such asreceptor-mediated endocytosis, or by non-specific means such aspinocytosis

The practice of the present application may employ conventional methodsof virology, immunology, microbiology, molecular biology and recombinantDNA techniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of the presentapplication can be administered in the form of pharmaceuticalcompositions. These compositions can be prepared in a manner well knownin the pharmaceutical art, and can be administered by a variety ofroutes, depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration routes include, but arenot limited, to pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal orintranasal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal intramuscularor injection or infusion; or intracranial, e.g., intrathecal orintraventricular, administration. Parenteral administration can be inthe form of a single bolus dose, or may be, for example, by a continuousperfusion pump. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

This application also includes pharmaceutical compositions whichcontain, as the active ingredient, the compound of the presentapplication or a pharmaceutically acceptable salt thereof, incombination with one or more pharmaceutically acceptable carriers(excipients). In some embodiments, the composition is suitable fortopical administration. In making the compositions of the presentapplication, the active ingredient is typically mixed with an excipient,diluted by an excipient or enclosed within such a carrier in the formof, for example, a capsule, sachet, paper, or other container. When theexcipient serves as a diluent, it can be a solid, semi-solid, or liquidmaterial, which acts as a vehicle, carrier or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The compounds of the present application may be milled using knownmilling procedures such as wet milling to obtain a particle sizeappropriate for tablet formation and for other formulation types. Finelydivided (nanoparticulate) preparations of the compounds of the presentapplication can be prepared by processes known in the art, e.g., seeInternational App. No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the present application can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the compositions of the present application containfrom about 5 to about 50 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodiescompositions containing about 5 to about 10, about 10 to about 15, about15 to about 20, about 20 to about 25, about 25 to about 30, about 30 toabout 35, about 35 to about 40, about 40 to about 45, or about 45 toabout 50 mg of the active ingredient.

In some embodiments, the compositions of the present application containfrom about 50 to about 500 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodiescompositions containing about 50 to about 100, about 100 to about 150,about 150 to about 200, about 200 to about 250, about 250 to about 300,about 350 to about 400, or about 450 to about 500 mg of the activeingredient.

In some embodiments, the compositions of the present application containfrom about 500 to about 1000 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodiescompositions containing about 500 to about 550, about 550 to about 600,about 600 to about 650, about 650 to about 700, about 700 to about 750,about 750 to about 800, about 800 to about 850, about 850 to about 900,about 900 to about 950, or about 950 to about 1000 mg of the activeingredient.

Similar dosages may be used of the compounds described herein in themethods and uses of the present application.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present application. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present application.

The tablets or pills of the present application can be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermit the inner component to pass intact into the duodenum or to bedelayed in release.

A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

The liquid forms in which the compounds and compositions of the presentapplication can be incorporated for administration orally or byinjection include aqueous solutions, suitably flavored syrups, aqueousor oil suspensions, and flavored emulsions with edible oils such ascottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.

Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face mask, tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of a compound of the present application can varyaccording to, for example, the particular use for which the treatment ismade, the manner of administration of the compound, the health andcondition of the patient, and the judgment of the prescribing physician.The proportion or concentration of a compound of the present applicationin a pharmaceutical composition can vary depending upon a number offactors including dosage, chemical characteristics (e.g.,hydrophobicity), and the route of administration. For example, thecompounds of the present application can be provided in an aqueousphysiological buffer solution containing about 0.1 to about 10% w/v ofthe compound for parenteral administration. Some typical dose ranges arefrom about 1 μg/kg to about 1 g/kg of body weight per day. In someembodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kgof body weight per day. The dosage is likely to depend on such variablesas the type and extent of progression of the disease or disorder, theoverall health status of the particular patient, the relative biologicalefficacy of the compound selected, formulation of the excipient, and itsroute of administration. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Synthesis

Compounds of the present application, including salts thereof, can beprepared using known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes, for example, bymethods analogous to those described in the Examples section.

The reactions for preparing compounds of the present application can becarried out in suitable solvents which can be readily selected by one ofskill in the art of organic synthesis. Suitable solvents can besubstantially non-reactive with the starting materials (reactants), theintermediates, or products at the temperatures at which the reactionsare carried out, e.g., temperatures which can range from the solvent'sfreezing temperature to the solvent's boiling temperature. A givenreaction can be carried out in one solvent or a mixture of more than onesolvent. Depending on the particular reaction step, suitable solventsfor a particular reaction step can be selected by the skilled artisan.

Preparation of compounds of the present application can involve theprotection and deprotection of various chemical groups. The need forprotection and deprotection, and the selection of appropriate protectinggroups, can be readily determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3^(rd)Ed., Wiley & Sons, Inc., New York (1999), which is incorporated hereinby reference in its entirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC), liquid chromatography-mass spectroscopy(LCMS), or thin layer chromatography (TLC).

Methods on how to prepare optically active forms from optically inactivestarting materials are known in the art, such as by resolution ofracemic mixtures or by stereoselective synthesis. Many geometric isomersof olefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present application. Cis and trans geometric isomers of thecompounds of the present application are described and may be isolatedas a mixture of isomers or as separated isomeric forms.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An example method includes fractionalrecrystallizaion using a chiral resolving acid which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids such asβ-camphorsulfonic acid. Other resolving agents suitable for fractionalcrystallization methods include stereoisomerically pure forms ofα-methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Kits

The present application also includes pharmaceutical kits useful, forexample, in the treatment or prevention of any of the disease statesdescribed herein, which include one or more containers containing apharmaceutical composition comprising a therapeutically effective amountof a compound of the present application. Such kits can further include,if desired, one or more of various conventional pharmaceutical kitcomponents, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

EXAMPLES

The methods and compounds will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnon-critical parameters which can be changed or modified to yieldessentially the same results.

Example 1.N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine

4-Methoxybenzhydrazide (0.332 mg, 2.0 mmol) was dissolved inacetonitrile (10 mL) and to this mixture was added1,1′-thiocarbonyldiimidazole (445 mg, 2.5 mmol). After stirring at rtfor 3 hours, 2-amino-4-methoxy-benzothiazole (450 mg, 2.5 mmol) wasadded and the temperature raised to 85° C. with stirring continued for20 hours. After cooling, the acetonitrile was removed by rotaryevaporator. Water was added (100 mL) and this mixture was extracted with3 volumes of ethyl acetate (50 mL). The organic layers were collectedand solvent removed en vacuo. To this thiosemicarbazide intermediate wasadded tosyl chloride (393 mg, 2.1 mmol) and pyridine (0.29 mL, 3.6 mmol)in THF (20 mL). The solution was heated to 70° C., bringing the mixtureto reflux for 20 h, then cooled. Ethyl acetate (10 mL) and HCl (1.0 M,10 mL) were added and the mixture was vigorously stirred for 10 minutes.The aqueous layer was removed and extracted with EtOAc (20 mL), and thecombined organic layers were flushed with concentrated to a slurry.After purification via reverse-phase HPLC (water/acetonitrile, 10-95%),the product fractions were collected, concentrated and lyophilized tofurnish a white solid. ESI-MS m/z 355 [M+H]⁺.

The following Examples 2-18 were purchased from Life Chemicals Inc.,Ontario, Canada.

Example 2.N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine

Example 3.N-(4-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 4.N-(4-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 5.5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine

Example 6.5-(2,4-dimethoxyphenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine

Example 7.N-(4-fluorobenzo[d]thiazol-2-yl)-5-phenyl-1,3,4-oxadiazol-2-amine

Example 8.N-(6-methoxybenzo[d]thiazol-2-yl)-5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 9.N-(6-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 10.5-(4-chlorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine

Example 11.5-(2-chlorophenyl)-N-(6-fluorobenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine

Example 12.N-(6-fluorobenzo[d]thiazol-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine

Example 13.N-(6-fluorobenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 14.N-(6-fluorobenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 15.N-(5-methoxybenzo[d]thiazol-2-yl)-5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 16.N-(5-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 17.N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine

Example 18.5-(4-methoxyphenyl)-N-(6-(methylthio)benzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine

Materials and MethodsAntibodies and Transfection

The following antibodies were used for immunoblotting andimmunoprecipitation (IP): polyclonal anti-SERCA2a (21th century BiochemInc.), monoclonal anti-GAPDH (Sigma-Aldrich, catalog no. G8795),monoclonal anti-SUMO 1 (Cell Signaling Technology Inc., catalog no.4930), anti-mouse horseradish peroxidase (HRP) (Pierce BiotechnologyInc., catalog no. 32430), and anti-rabbit HRP (Pierce BiotechnologyInc., catalog no. 32460). YFP-tagged SUMO1, pcDNA3.0-SERCA2a, andHA-tagged Ubc9 plasmids were used for the transfection. The plasmid DNAwas amplified in the Escherichia coli strain DH5a and extracted by usinga commercial purification kit (Qiagen, catalog no. 12263). Purifiedplasmid was resuspended in sterile TE buffer (10 mM Tris-HCl and 1 mMEDTA, pH 7.6). Only the preparation highest purity (A260/A280>1.8) wasused for transfection. 1 μg of each plasmid was used for transfection.HEK-293 cells (American Type Culture Collection, catalog no. CRL-1873)were grown at 37° C. and under a 5% CO2 humidified atmosphere inDulbecco's modified Eagle's medium (DMEM, Cellgro, catalog no. 10-013-CM) containing 10% fetal bovine serum (SAFC Bioscience, catalog no.12107) and 100 i.u of penicillin/ml and 100 μg of streptomycin/ml.HEK-293 cells were seeded at a density of 3-5×10⁵ cells per 60 mmculture dish in DMEM. The cells were transiently transfected usingLipofectamine 2000 (Invitrogen, catalog no. 11668) with indicatedexpression plasmids. After 24 hours, the cells were treated with eithersmall molecules or dimethyl sulfoxide (DMSO, Sigma-Aldrich, catalog no.D2650).

Cardiac Myocyte Isolation

Calcium-tolerant adult rat ventricular myocytes (ARVMs) were obtainedfrom hearts of male Sprague-Dawley rats (250 to 300 g). The heart wasexcised and perfused with a standard enzymatic technique. ARVMs wereplated on multi-chambered plates or culture dishes, pre-coated withlaminin 2 μg/cm², at a density of 10⁵ cells/cm² in DMEM withoutL-glutamine supplemented with 10 mmol/l HEPES, 3.7 mg/ml NaHCO₃, 1 mg/mlglucose, 0.11 mg/ml sodium pyruvate, 2 mg/ml bovine serum albumin, 2mmol/1 L-camitine, 5 mmol/1 creatine, 5 mmol/1 taurine, 1%penicillin-streptomycin, and 1% gentamycin. Following isolation, cellswere allowed to settle for 1 hour. Cultures were incubated at 37° C., inan atmosphere of 5% CO₂₋₉₅% air. Fresh medium was added gently as mediumwas being drawn off until the cultures had been thoroughly washed. Onlyquiescent, rod-shape cardiac myocytes were selected for IonOptixexperiments.

Example Compounds

Each compound was added at 10 μM. DMSO was used as a control. After 24hours at 37° C., SERCA2a SUMOylation and functional analysis weredetermined.

Cell Image

Cellular permeability and potential activity of the compounds wereexamined by tracking of the YFP-SUMO1. HEK-293 cells expressingYFP-SUMOI and pcDNA3.0-SERCA2a were incubated with either 10 μM DMSO or10 μM small molecules for 24 hours at 37° C. Fluorescent signals weremonitored by fluorescence microscopy. HA-tagged Ubc9 expressing cellswere served as a positive control.

Cell Shortening/Re-Lengthening

Mechanical properties of ARVMs were assessed using an IonOptix MyoCam®system (IonOptix, Milton, Mass.). In brief, cells were placed in aWarner chamber mounted on the stage of an inverted microscope (Olympus,IX-70) and superfused (1 ml/min at 30° C.) with a buffer containing 131mM NaCl, 4 mM KCl, 1 mM CaCh, 1 mM MgCh, 10 mM glucose, and 10 mM HEPES,pH 7.4. ARVMs were field stimulated with suprathreshold voltage and at afrequency of 0.5 Hz. The ARVMs being studied was displayed on thecomputer monitor using an IonOptix MyoCam camera. SoftEdge software(IonOptix) was used to capture changes in cell length during shorteningand re-lengthening.

Intracellular Fluorescence Measurement

ARVMs were placed in a chamber on an Olympus IX-70 inverted microscopeand imaged through a Fluor 40× oil objective. ARVMs were exposed tolight emitted by a 75 W lamp and passed through either a 360 or a 380 nmfilter (bandwidths were ±15 nm), while being stimulated to contract at0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by aphotomultiplier tube after first illuminating cells at 360 nm for 0.5 sthen at 380 nm for the duration of the recording protocol (333 Hzsampling rate). The 360 nm excitation scan was repeated at the end ofthe protocol and qualitative changes in intracellular Ca²⁺ concentration((Ca²⁺]_(i)) were inferred from the ratio of the fluorescence intensityat two wavelengths.

Immunoblotting

Equal amounts of protein from either small molecule treated or DMSOtreated cells or immunoprecipitates were resolved by 7.5% SDS-PAGE andtransferred to nitrocellulose membranes (Bio-Rad, catalog no. 162-0112).The membranes were blocked for 1 hour at room temperature with 5%non-fat milk (Cell Signaling Technology Inc., catalog no. 9999) in TBST(10 mM Tris-HCl, 150 mM NaCl, and 0.05% tween-20, pH 8.0). The blotswere incubated with specific primary antibodies at 4° C. for overnight.The blots were then washed five times for 10 minutes each with TBST andincubated for 1 hour with HRP-conjugated secondary antibodies in TBSTwith 5% non-fat milk. After five times TBST washes, the protein bandswere visualized with enhanced chemiluminescence (Pierce BiotechnologyInc., catalog no. 32132) and exposed to x-ray film (Denville ScientificInc., catalog no. E3012). GAPDH expression provided an internal control.

SERCA2a SUMOylation Assay

Post-transfection (48 hours), the HEK-293 cells were rinsed twice withphosphate-buffered saline (PBS, Cellgro, catalog no. 21-040-CM) andlysed in 1% Nonidet P-40 lysis buffer (Boston Bioproducts, catalog no.BP-119) with 10 mM N-ethylmaleimide (NEM, Sigma-Aldrich, catalog no.N3876) and phosphatase inhibitor cocktail (Complete Mini Tablet, RocheApplied Science, catalog no. 11836153001). 2 mg of protein were mixedwith the anti-SERCA2a antibodies for overnight at 4° C. in lysis buffer.Pre-washed protein A-Separose beads (Pierce Biotechnology Inc., catalogno. 20333) was added to each sample and incubated 1 hour at 4° C. withgentle rocking. Immunocomplexes were washed with lysis buffer threetimes and precipitated by centrifugation at 12000×g for 10 seconds. Theimmunocomplexes were resuspended in SDS sample buffer and subjected toimmunoblotting. Controls for the immunoprecipitations were performedusing an anti-rabbit IgG equal to that of the primary precipitatingantibody. Ten percent of whole cell lysates used in theimmunoprecipitation was loaded for subsequent immunoblotting.

Statistics

Data were obtained from experiments performed two or three times andvalues are presented as mean±standard deviation (SD). The p value wascalculated by analysis of variance, followed by Student's t test.Difference between the groups of data were considered statisticallysignificant when p<0.05.

Compound Screening Data

Referring to FIG. 7, thirty small molecules were screened for theirability to SUMOylate SERCA2a in HEK-293 cells at 10 μM concentration.Fourteen non-cytotoxic compounds were used. Referring to FIG. 7b ,cellular activity of the compounds was confirmed by using cell-imagingstep. Of the fourteen compounds tested, referring to FIG. 7c , eightcompounds were selected based on their effect on increasing SERCA2aSUMOylation in HEK-293 cells. The mechanical properties of the eightcompounds were assessed by using IonOptix system. Referring to FIG. 8a ,four of these compounds were found to increase cell contractility andtau significantly (p<0.05). Referring to FIG. 8b , endogenous SERCA2aSUMOylation status was also determined in the same set of cells. Onlytwo compounds showed both positive inotrophic effect and enhancement ofendogenous SERCA2a SUMOylation.

Example A: Induction of YFP-SUMO1 Accumulation and SERCA2A SUMOylationin HEK-293 Cells

The ability of the Example compound to SUMOylate SERCA2a was examinedusing an in vitro system in HEK 293 cells where both SERCA2a and SUMO 1are expressed. The cellular permeability and potential activity of theExample compounds was examined by tracking of the YFP-SUM01. HEK-293cells expressing YFP-SUM01 and pcDNA3.0-SERCA2a were treated with either10 μM DMSO or 10 μM indicated Example 1 for 24 hrs. Fluorescent signalswere monitored by fluorescence microscopy. HA-Ubc9 expressing cells wereserved as a positive control. The results are shown in FIG. 1A (scalebars, 10 μM). The SERCA2a SUMOylation profiling was then analyzed byusing the same cells (FIG. 1B, WCL, whole cell lysates; S-SERCA2a,SUMOylated SERCA2a).

Example B: Effect of Example 1 on Intracellular Calcium Decay, CellContractility and SERCA2a SUMOylation in Isolated Adult Cardiomyocytes

The effects of the Example compound was examined on isolated ratcardiomyocytes shortening and relaxation parameters. Example 1 showedpositive inotropic and lusitropic effects on cardiac function. Effectsof Example 1 on mechanical properties of the ARVMs were assessed using avideo-based edge detection system (IonOptix). The time constant of theincrease of force during the stretch decreased (tau), percentage of peakshortening (PS), maximal rate of relaxation and contraction wereanalyzed at 24 hrs postincubation of the compounds in culture media(N=10 ARVMs). The mechanical data were analyzed with student's t-test(FIG. 2A, P<0.05 compared to the DMSO treated cells). SERCA2aSUMOylation profiling was analyzed in a same set of ARVMs (FIG. 2B, WCL,whole cell lysates; S-SERCA2a, SUMOylated SERCA2a).

Example C: Effect of Example 1 on SERC2a Function

Treatment with Example 1 significantly increased the rate of transientcalcium decay (Tau (msec); Example 1, 1.8±0.2; DMSO, 2.4±0.2, p<0.05),and cell contraction (Peak shortening (%); Example 1, 12.0±0.8, DMSO,3.0±0.8; Maximal rate of contraction (μm/sec); Example 1, −185.5±6.5,DMSO, −64.0±7.9; Maximal rate of relaxation (μm/sec); Example 1,210.7±19.4, DMSO, 54.0±2.4, p<0.001, n=10) and endogenous SERCA2aSUMOylation in isolated rodent adult cardiomyocytes (FIG. 8a ). Tofurther validation the efficiency of Example 1 for cardiac function, weperformed dose response experiments. The results indicated that Example1 significantly increased cell contractility (peak shortening (%);p<0.05, versus DMSO and 10 μM; p<0.001, versus DMSO) in a concentrationdependent manner. Tau value, which is an indicator of SERCA2a pumpfunction, was also significantly decreased with an increasing amount ofExample 1 (0.1 μM Example 1, 1.3±0.0 msec, 1 μM Example 1; 1.3±0.1 sec,p<0.05, versus DMSO and 10 μM Example 1, 1.0±0.1 msec, p<0.001, versusDMSO) (FIG. 3b , left). In addition, increasing Example 1 increasedlevels of SERCA2a SUMOylation (FIG. 3b , right). However, the proteinexpression levels of E1, E2 enzymes, SERCA2a and SUMO-1 were notchanged.

Example D: Effect of Example 1 on the SUMO Activating Enzyme, E1

Example 1 activated ATP-dependent activation of SUMO-1 (EC₅₀=40.47±4.4μM, FIG. 4a ) and thioester formation between the SUMO-1 and SUMO E1enzyme (EC₅₀=1.85±0.96 μM, FIG. 4b ) and subsequent transfer of theactivated SUMO-1 from SUMO E1 to the unique SUMO E2 enzyme, UBC9(EC₅₀=10.17±0.28 μM, FIG. 4c ) in a gel-based assay. To betterunderstand the structural basis for the action of Example 1 on SUMO E1enzyme, in silico modeling was also performed (FIG. 4d ).

Example E: Effect of Example 1 on Contractility

Hemodynamics were measured to evaluate the acute effect of Example 1infusion on the cardiac function of the mouse model of HF. In the firstset of experiments, mice were exposed to pressure overload by thoracicaortic constriction (TAC) or sham operation and received either EXAMPLE1 or vehicle. At two months post-TAC animals developed HF. Leftventricle (LV) was severely dilated and the cardiac function asdetermined by fractional shortening and ejection fraction, weresignificantly decreased. The sham-operated animals showed nostatistically significant changes in echocardiographic parameters. TheSERCA2a protein levels were significantly decreased by 40% of the normallevel in TAC mice. In addition, the positive chronotropic effect ofdobutamine was no longer observed in TAC mice (FIG. 9). Followingcannulation of the carotid artery and external jugular vein, infusion ofEXAMPLE 1 or vehicle was performed as described in Method section.Pressure and conductance catheters were introduced in the LV through thecarotid artery. Pressure and volume measurements were madesimultaneously and pressure volume curves were constructed as thevehicle or EXAMPLE 1 were infused.

Hemodynamic data were obtained in 1 mg of EXAMPLE 1-treated failinghearts at 30 min and 60 min after compound treatment. The end systolicpressure-volume relationship (ESPVR) in the LV was significantly steeperin EXAMPLE 1 treated mice than baseline (p<0.001) (FIG. 9a ). Inaddition, the rate of LV pressure (dP/dt max) rise in EXAMPLE 1 treatedmice compared to the baseline (p<0.001), suggesting increased cardiaccontractility. The improvement occurred within 30 minutes aftertreatment and maintained for 60 minutes after treatment. Moreimportantly, infusion of EXAMPLE 1 induces an increase in the index ofcontractility, ESPVR in a dose dependent manner. Higher doses of EXAMPLE1 induced a higher increase in ESPVR. The effects of EXAMPLE 1 onvarious hemodynamic parameters are shown in FIG. 9b and Table S1. dP/dtmax increased with increasing concentration of EXAMPLE 1 while Tau aparameter of relaxation decreased an indication of enhanced relaxation.The vehicle infusion induces no change in the pressure volumerelationship and does not affect the index of contractility, ESPVR anddP/dt max in both sham and TAC mice (FIG. 10a ). No statisticallysignificant changes in hemodynamic parameters were observed insham-operated groups treated with EXAMPLE 1 (FIG. 10b ).

TABLE S1 Hemodynamic parameters in 2 months after TAC animals treatedwith Example 1. TAC + Example 1 (n = 5 each group) Parameters Baseline0.1 mg/kg 1.0 mg/kg 10 mg/kg ESPVR slope 3.1 ± 9.0 ± 11.2 ± 20.3 ±(mmHg/A) 0.3 0.8* 3.1* 1.3* +dP/dt Max 4738.7 ± 4803.3 ± 5448.8 ± 6229.4± (mmHg/sec) 284.1 290.2 465.1* 357.6* Pmax 111.1 ± 115.1 ± 123.6 ±124.5 ± (mmHg) 6.4 4.4 18.3* 14.7* Tau(1/2) 7.7 ± 7.5 ± 6.4 ± 6.2 ± 0.4*(msec) 0.4 0.5 0.5* HR 422.1 ± 417.1 ± 422.3 ± 455.1 ± (bpm) 35.5 15.713.1 19.4* ESPVR (end systolic pressure volume relationship), +dP/dt Max(peak rate of pressure rise), Pmax (maximal pressure), Tau(1/2)(relaxation time constant), HR (heart rate) in mice subjected to TAC(transverse aorta constriction) with different dose of the Example 1.Data are given as means ± SD. *, P < 0.05 by Student's t-test, versusBaseline.

Example F. Effects of Example 1 on Serca2 Knockout Mice

Adult mice with an inducible cardiomyocyte-specific excision of theAtp2a2 (Serca2) gene (Serca2 KO) were used. Four weeks after inductionof Serca2 gene excision, the mice display a substantial reduction insystolic and diastolic function (FIG. 11a ). Ejection fraction wassignificantly lower in Serca2 KO mice compared with wild-type (WT)control (Serca2 KO, 80.90±0.23%; WT, 95.4±0.23%, p<0.001). LV fractionalshortening was significantly decreased in Serca2 KO mice (Serca2 KO,43.80±9.16%; WT, 65.04±0.59%, p<0.001). LV dimension, as measured byLVIDd and LVIDs, were significantly (p<0.001) increased. The decayconstant, tau value was nearly 1.3-fold (Serca2 KO, 9.66±0.57 sec; WT,7.33±0.5 sec, p<0.001) larger in Serca2 KO mice compared with WT control(FIG. 11b ). The expression of SERCA2a was completely lacking in Serca2KO mice (FIG. 11c ).

Hemodynamic parameters obtained before and after infusion of Example 1were compared. As shown in FIG. 6, Example 1 did not induce anyimprovements in hemodynamic parameters in Serca2 KO mice. ESPVR slope(p=0.17, versus baseline), dP/dt max (p=0.28. versus baseline), Pmax(p=0.66 versus baseline), Tau (p=0.10 versus baseline), and heart rate(p=0.06 versus baseline) were not statistically significant difference(FIG. 6a ). The dose-dependent enhancement of cardiac contractilitydisappeared in Serca2 KO mice (FIG. 6b ).

Example G. Pharmacokinetic Profile of Example 1

In a murine model, the half-life of Example 1 is 65.4 minutes with aCmax of 2.24 μM (FIG. 12).

Example H. Cancer Cell Line Screen for Example 1

The NCI-60 human tumor cell screen used by the National CancerInstitute's Developmental Therapeutics Program was performed onExample 1. The NCI-60 set includes leukemia, lymphomas, and carcinomasof ovarian, renal, breast, prostate, colon, lung and CNS origin. Theresults of the screen revealed that Example 1 did not induce cancer cellgrowth and proliferation (FIG. 13).

Example I. Effect of Examples 1-4 on SUMOylation

Referring to FIG. 14, Examples 1-4 induce accumulation of SUMO1 in thenucleus indication of a positive screen. Examples 1-4 also induceSERCA2a SUMOylation in HEK 293 cells.

Example J. Effect of Example 1 at Different Doses on SUMOylation

Referring to FIG. 15, Example 1 increases SUMOylation of SERCA2a in adose dependent manner.

Example K. Effect of Example 1 in a Mouse Heart Failure Model

A model of heart failure was created by transaortic constriction in themouse for 6-8 weeks. Following cannulation of the carotid artery andexternal jugular vein, infusion of Example 1 or vehicle was performed asshown in the protocol in FIG. 16. Pressure and conductance catheterswere introduced in the left ventricle through the carotid artery.Pressure and volume measurements were made simultaneously and pressurevolume curves were constructed as the vehicle or Example 1 was infused.

As shown in FIG. 17, vehicle infusion induces no change in the pressurevolume relationship and does not affect the index of contractilityend-systolic pressure volume relationship (ESPVR).

As shown in FIG. 18, infusion of Example 1 induces an increase in theindex of contractility ESPVR in a dose dependent fashion. Higher dosesof Example 1 induced a higher increase in ESPVR.

The effects of Example 1 on various hemodynamic parameters are shown inFIG. 19. Example 1 increased various hemodynamic parameters in aconcentration dependent fashion. dP/dtmax increased with increasingconcentration of Example 1 while Tau, a parameter of relaxation,decreased, an indication of enhanced relaxation.

Example L. Effect of Example 1 in a Non-Rodent Heart Failure Model

Example 1 was dosed in Yorkshire pigs at 5 mg/kg with severe heartfailure secondary to myocardial infarction. Referring to FIG. 20,infusion of Example 1 induced an increase in contractility (as measuredby dP/dt) by 25-30%.

Experimental Protocol for Pig Study

Animal

The experimental protocols complied with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals and standards ofUnited States regulatory agencies. They were approved by theInstitutional Animal Care and Use Committee of the Mount Sinai School ofMedicine. Yorkshire pigs were pre-medicated using intramuscular Telazol™(8.0 mg/kg; tiletamine hydrochloride and zolazepam hydrochloride; FortDodge Animal Health, Fort Dodge, Iowa). After the placement of anintravenous line, animals were intubated and ventilated with 100%oxygen. General anesthesia was maintained with intravenous propofol(8-10 mg/kg/hr) throughout the procedure.

Myocardial Infarction (MI) Creation

The animals underwent a 2-hour balloon occlusion of the proximal LAD(left anterior descending coronary artery) followed by reperfusion. Tenminutes before MI creation a continuous infusion of 20 mEq potassiumacetate, 75 mg amiodarone, and 2 mg atropine was initiated to reduceischemia related arrhythmia. In addition, a 75-mg bolus of amiodaronewas administered i.m. Then, the left coronary artery was accessed with a7 French hockey stick catheter (Cordis), and a 4-mm over-the-wireVOYAGER angioplasty catheter (Abbott) was advanced into the proximalLAD. After confirming the correct position, the balloon was inflated to4 atm. Arterial pressure, blood oxygen saturation (SpO₂), andelectrocardiogram were monitored closely throughout the procedure. Theanimals were defibrillated with 200 J, if necessary, and arterialhypotension was corrected with rapid i.v. saline infusion, if required.

Cardiac Performance Assessment

Cardiac performance was evaluated at one month after MI. A 7 Frenchhockey stick catheter (Cordis) was advanced to the left coronary artery.After the coronary angiogram, a 0.014-inch guide wire (Abbott) wasadvanced into the LAD and 8-mm-long, 4.0-mm VOYAGER over-the-wireballoon (Abbott) was advanced to the proximal part of the coronaryartery. The balloon was then inflated to 3-4 atm for 120 minutesfollowed by reperfusion. Example 1 (5 mg/kg) was administered byintracoronary injection (¾ to the left coronary artery and ¼ to theright coronary artery). Cardiac function was monitored and recorded for3 hours after injection.

Additional embodiments of Formula (II) were tested for effects onSERCA2a SUMOylation and cardiac contractility. The results aresummarized in Table 2.

TABLE 2 Cardiac effects of some embodiments of Formula (II). Solubility/YFP SERCA2a Cardiac ID Structure Toxicity accumulation SUMOylationContraction Example 2

10 mM in DMSO/ Non-toxic Active (2.93x increase vs. DMSO) Increased1.34x compared with DMSO control Peak shortening 2.2x compared to DMSOcontrol Example 3

10 mM in DMSO/ Non-toxic Active (2.49x increase vs. DMSO) Increased1.43x compared with DMSO control Peak shortening 2.5x compared to DMSOcontrol Example 4

10 mM in DMSO/ Non-toxic Active (2.34x increase vs. DMSO) Increased 1.4xcompared with DMSO control Peak shortening 4.3x compared to DMSO controlExample 5

10 mM in DMSO/ Non-toxic Active (2.54x increase vs. DMSO) Notstatistically significant — Example 6

10 mM in DMSO/ 30% Active (2.93x increase vs. DMSO) Not statisticallysignificant — Example 7

10 mM in DMSO/ Non-toxic Active (2.2x increase vs. DMSO) Notstatistically significant — Example 8

10 mM in DMSO/ 30% Active (2.37x increase vs. DMSO) Not statisticallysignificant — Example 9

Crystallized at 2.5 mM/ Toxic Active Not statistically significant —Example 10

Solubility <2.5 mM/ >50% Active Not statistically significant — Example11

Solubility <2.5 mM/ >50% Active Not statistically significant — Example12

Solubility <2.5 mM/ >50% Active Not statistically significant — Example13

Solubility <2.5 mM/ >50% Active Not statistically significant — Example14

Solubility <2.5 mM/ >50% Active Not statistically significant — Example15

Solubility <2.5 mM/ >50% Active Not statistically significant — Example16

Solubility <2.5 mM/ >50% Active Not statistically significant — Example17

5 mM/ Non-toxic Active (2.2x increase vs. DMSO) Not statisticallysignificant — Example 18

Solubility <2.5 mM/ >50% Active Not statistically significant —

What is claimed is:
 1. A method of treating heart failure, cardiachypertrophy, myocarditis, myocardial infarction, ischemia, cardiacarrhythmias, vascular rhexis, cardiac arrhythmia, valvulopathy,diastolic dysfunction, hypertension, neurodegenerative disorders, viralinfection, liver disease, or inflammation in a patient in need thereof,comprising administering to said patient a therapeutically effectiveamount of a compound of Formula II:

or a pharmaceutically acceptable salt thereof; wherein: Ar is aryl orheteroaryl, each of which is optionally substituted with 1, 2, 3, 4, 5,or 6 independently selected R^(1a) groups; W is S or O; Y is O; each Xis N; Z is O, S, or NR^(A); R^(A) is H or C₁₋₄ alkyl; R¹, R², R³, and R⁴are each independently selected from hydrogen, CN, nitro, hydroxy, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆alkylamino, di-C₁₋₄-alkylamino, carboxy, carbamyl, C₁₋₆ alkylcarbamyl,di(C₁₋₄alkyl)carbamyl, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆alkylsulfonylamino, aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di-C₁₋₄alkylamino sulfonyl, aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino,and di-C₁₋₄alkylaminosulfonylamino; wherein said C₁₋₆ alkyl, C₁₋₆haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylamino,di-C₁₋₄-alkylamino, C₁₋₆ alkylcarbamyl, di(C₁₋₄alkyl)carbamyl, and C₁₋₆alkylcarbonyl are each optionally substituted with 1, 2, or 3 groupsindependently selected from halo, CN, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃alkylamino, and di-C₁₋₃-alkylamino; each R^(1a) is hydrogen.
 2. A methodaccording to claim 1, wherein Ar is phenyl.
 3. A method according toclaim 1, wherein W is S.
 4. A method according to claim 1, wherein Z isNH.
 5. A method according to claim 1, wherein R¹, R², R³, and R⁴ areindependently selected from hydrogen, CN, nitro, hydroxy, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆ alkylamino,and di-C₁₋₄-alkylamino.
 6. A method according to claim 1, wherein R¹,R², R³, and R⁴ are independently selected from hydrogen and C₁₋₆ alkoxy.7. A method according to claim 1, wherein at least three of R¹, R², R³,and R⁴ are hydrogen.
 8. A method of treating heart failure, cardiachypertrophy, myocarditis, myocardial infarction, ischemia, cardiacarrhythmias, vascular rhexis, cardiac arrhythmia, valvulopathy,diastolic dysfunction, hypertension, neurodegenerative disorders, viralinfection, liver disease, or inflammation in a patient in need thereof,comprising administering to said patient a therapeutically effectiveamount of a compound of Formula II:

or a pharmaceutically acceptable salt thereof; wherein: Ar is aryl orheteroaryl, each of which is optionally substituted with 1, 2, 3, 4, 5,or 6 independently selected R^(1a) groups; W is S or O; Y is O; each Xis N; Z is O, S, or NR^(A); R^(A) is H or C₁₋₄ alkyl; R¹, R², R³, and R⁴are each hydrogen; each R^(1a) is independently selected from halo, CN,nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₇cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, carboxy,carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₄alkyl)carbamyl, C₁₋₆ alkylcarbonyl,C₁₋₆ alkoxycarbonyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl,C₁₋₆alklaminosulfonyl, di-C₁₋₄ alklaminosulfonyl, aminosulfonylaminoC₁₋₆ alkylaminosulfonylamino, and di-C₁₋₄alkylaminosulfonylamino;wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₇ cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆ alkylcarbamyl,di(C₁₋₄ alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are each optionallysubstituted with 1, 2, or 3 groups independently selected from halo, CN,hydroxy, C₁₋₃ alkoxy, amino, alkylamino, and di-C₁₋₃-alkylamino; or twoadjacent R^(1a) taken together with the atoms to which they are attachedcan form a 3-7 membered carbocyclic or 4-6 membered heterocyclic ring,each of which is optionally substituted with 1, 2, 3, or 4 C₁₋₃ alkylgroups.
 9. A method according to claim 8, wherein the compound is acompound of Formula Ia:

or a pharmaceutically acceptable salt thereof.
 10. A method according toclaim 8, wherein the compound is a compound of Formula Ib:

or a pharmaceutically acceptable salt thereof.
 11. A method according toclaim 8, wherein the compound is a compound of Formula Ic:

or a pharmaceutically acceptable salt thereof wherein: R^(2a), R^(2b),R^(2c), R^(2d), and R^(2e) are each independently selected fromhydrogen, halo, CN, nitro, hydroxy, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl,C₁₋₆ heteroaryl, C₁₋₆haloalkoxy, amino, C₁₋₆alkylamino, carboxy,carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₄alkyl)carbamyl, C₁₋₆ alkylcarbonyl,C₁₋₆alkoxycarbonyl, C₁₋₆alkylcarbonyloxy, C₁₋₆alkylsulfonyl,C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl,C₁₋₆alkylaminosulfonyl, di-C₁₋₄ alkylaminosulfonyl, aminosulfonylamino,C₁₋₆alkylaminosulfonylamino, and di-C₁₋₄ alkylaminosulfonylamino;wherein said C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₇cycloalkyl, C₂₋₆ heterocycloalkyl, phenyl, C₁₋₆ heteroaryl, C₁₋₆haloalkoxy, C₁₋₆ alkylamino, di-C₁₋₄-alkylamino, C₁₋₆alkylcarbamyl,di(C₁₋₄alkyl)carbamyl, and C₁₋₆ alkylcarbonyl are each optionallysubstituted with 1, 2, or 3 groups independently selected from halo, CN,hydroxy, C₁₋₃ alkoxy, amino, C₁₋₃ alkylamino, and di-C₁₋₃-alkylamino.12. A method according to claim 11, wherein R^(2a), R^(2b), R^(2c),R^(2d), and R^(2e) are each independently selected from hydrogen, halo,CN, hydroxy, C₁₋₆ alkyl, C₁₋₆ haloalkyl, and C₁₋₆ haloalkoxy.
 13. Amethod of treating heart failure, cardiac hypertrophy, myocarditis,myocardial infarction, ischemia, cardiac arrhythmias, vascular rhexis,cardiac arrhythmia, valvulopathy, diastolic dysfunction, hypertension,neurodegenerative disorders, viral infection, liver disease, orinflammation in a patient in need thereof, comprising administering tosaid patient a therapeutically effective amount of 5-(4chlorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine,or a pharmaceutically acceptable salt thereof.
 14. A method according toclaim 1, wherein said heart failure is selected from congestive heartfailure (CHF), chronic heart failure, and ischemic heart failure.
 15. Amethod according to claim 8, wherein the compound is a compound ofFormula IIc:

or a pharmaceutically acceptable salt thereof.
 16. A method according toclaim 8, wherein the method further comprises administering to thepatient an adeno-associated vector (AAV) comprising SERCA2a.